Auxin autotrophy and hyperauxinity of Eriophyes induced Zizyphus stem galls in Culture

Biochem. Physiol. Pflanzen 175, 537 -541 (1980)

Auxin Autotrophy and Hyperauxinity of Eriophyes Induced Zizyphus Stern GaUs in Culture P. TANDON and H. C. ARYA Department of Botany, School of Life Sciences, ~orth-Eastern Hill University, Shillong 793014, India

K e y Ter m 1nde x: ga.1I tissue f:ulture, growth regulators, growth, auxin autotrophy, hyperauxinity;

Zizyphus jujuba.

Summary In contrast to Zizyphus normal stem tissue, the gaU tissue was able to grow on MS medium free of auxin ami kinetin. With DL-tryptophan, both the tissues showed optimum growth at 5 mg/I, but the growth response of gaU tissue was much higher. The optimal concentration of NAA and IAA was 10 mg/I for growth of both normal and gall tissues. In vivo, and in vitra grown gaU tissues showed higher auxin contents as eompared to normal tissues. The endogenous auxin level in both gaU and normal tissues was highest in in vivo tissues, followcd by DL-tryptophan, XAA, and rAA treatments in decrea.sing order.

Introduction

The physiological autonomy shown by crown-gall tumor cell reflects an abnormal hormone metabolism (GAeTHERET 1947; BRAUN and STONIER 1958; STONIER 1969; MEINS 1974; ARYA ct al. 1975). It is now generally accepted that crown-gall tumor cells show auxin autotrophy and hyperauxinity (STONIER 1972; BUTcHER 1973; MEINS 1974). The nutritional autonomy of the crown-gall tissue could be due to cither change in requirements of the gaU tissue growth, or increased synthesis and retention of growth regulators. The literature is fuB of conflicting rcports on this point (STONIER 1972). In contrast to non-sclf-limiting tumor tissues in plants withetiological factors like bacteria, viruses, and genetic constitution, insect- und mite-induccd galls are reported to be self-Jimiting and continucd presence of insccts or mites is considered essential for gaB tissue growth (BRAUN 1969). Howcver, we reported ealier (TANDO~ et al. 1976) that galls induced by a plant mite, Eriophyes cemuus, on Zizyphus jujuba is of non-self-limiting type and the tissues possess tumcfacient properties. In an attcmpt to understand the abnormal growth hormone mctabolism of Zizyphus gall tissuc, the present studies on auxin contents in in vivo gaU and normal tissues, growth responses and auxin levels in both normal and gaU tissues, subjected to three growth regulators, werc carried out. Material and Methods Zizyphus jujuba LDIK. normal stem , stem galls incited by Eriophyes cenlUus MASSEE and tissue cultures of the same were llsed as experimental materia.l. Both norma.l aml gall tissues were isolated and maintained on ~lrRA S HI GF. and SKOOG (19G:2) medium as des cribed earlier (TANDON 1976). Abbreviations: medium.

~AA,

a-n,1phthalcneacetic acid; rAA, indole-3-acetic acid; MS, l\Iurashige-Skoog

538

P. T,\i'iDOl'i and H. C. AIlYA

The pH of the medium was adjusted to 5.8 before autoclaving it at 1.06 kg/sq.cm pressure for 20 min. The eultures were maintained through regular subculturing on M8 medium, and grown in dark growth eh amber maintained at 26 ± 2 °C and 50 to 60 % relative humidity. Growth of the tissues on M8 medium supplemented with 10 mg/I of NAA was used as contro!. Growth values(Final weight/initial weight) in M8 medium with different growth regulators are shown as percentage of contro!. The growth responses of normal and gall tissues to NAA, rAA, and DL-tryptophan weTe studied by incorporating different concentratiolls (0.1 to ~O mg/I) separately in auxin free 1\18 medium. Average growth responses of three r eplicate cultures for each treatment <1fter thirty days are presented as histograms (Figs. 1- 3). 8tlldies on tisslle growth and auxin contents (5th to 30th d<1Y <1t 5-d<1Y interval) were carried out in their third and fourth passa,ges of growth su bj ected to the above mentioned growth regul<1tors. rAA extradion a nd quantitative me<1surement in both in vivo and in vitro g<111 and normal tissues was done according to the method of MA HADEVAN (1975) which is basically as folIows: fresh tissues \Vere grou nd to <1 fine paste in a mortar with pestle, extraeted repeatedly with peroxide free ethyl ether, and then with 5 % sodium bicarbonate solution. The extract W<1S acidified to pU 3, and partiti oned in aseparatory funnel with ethyl ether. The ether extract was evaporated, dissolved in methyl aleohol, and separated by paper chromat ography using isopropanol-ammonia-water (80: 10: 10; v/v). A reference sampie of rAA was run simultaneously. The spot with the same Rf as that of known IAA was removed, eluted in 1 ml methyl alcohol. rAA content was determined by SALPER reagent using a sta.ndard cnrve, and expresscd as pg IAA equivalent per kg fresh weight of the tissue.

Results and Discussion During the present studios, it was found that normal and gaU tissues showed poor growth on auxin-kinetin free MS medium in the first passage of growth. hl subsequent passages, howevcr, normal tisses failed to grow, while the growth of gaU tissue increased considerably (Fig. 1). WHITE and BRAU N (1942) showed that bacteria-free crown-gall tissue cultures of sUllflower grew vigorously on a simple medium without growth substallces. These basic observations were subsequently confirmed and extended to crown-gaU tissues from other pl ant species ( B UTCHER 1973). The hybrid tumor tissue cultures derived from the tumor forming Nicotiana sauveolus x N. landsdorffii alld N. debneytabacurn X N. longiflora did not require auxin and kinetin for vigorous growth, whereas their non t umorous parents did (SCHAEFFER and Sl'vfITH 1963; AHUJA and HAGEN 1966). 100

-

! 80 :; ~ ~60 w

U

NORMAL

~ GALL ~

:::>

...J

~40 :r:

r

~

o

20

Ir C>

o

~~ 0.0

0·1 I 2·5 5-0 10-0 15·0 DL-TRYPTOPHAN mg/l IN MS MEDIUM

Auxin Autotrophy of Gall Tissue

539

100

DNORMAL

~GALL

o MEDIUM

Fig.2 100

D NORMAL ~ GALL

J: I~

o

20

0::

(9

o

Fig.3

5 I MS MEDIUM

The optimaI'concentration of NAA andIAA was 10 mgjl for growth of both the tissues. Above-optimal concentrations of NAA and IAA up to 20 mgjl in the medium, as shown in Figs. 2 and 3 respectively, showed steady growth of both normal and gaU tissues. For growth of both the tissues, NAA was found a superior source (Fig. 2), therfore, was considered control. This is consistant with the findings of PE LET et al. (1960) who found that NAA was a better source for the growth of Phylloxera gaU and normal stem tissues. During the present studies, effect of DIAryptophan on growth of Zizyphus gaU and normal tissues, grown on auxin-free medium showed that both the tissues utilized this compound with an optimal of 5 mgjl (Fig. 1). It was interesting to note that growth response of gaU tissue to DL-tryptophan was much higher as compared to normal tissue, and the gaU tissue showed more auxin content in this treatment (Table 1). BOUILLENE and GASPAR (1970) reported that the capacity of the crown-gall tissues of Impatiens to synthesize rAA like substances from tryptophan was particularly high. As shown in the table, from 5th to 30th day of growth, at 5-day interval, both in vivo and in vitro gaU tissues showed more auxin contents than the corresponding normal 37

Biochern. Physiol. Pllanzen, Bd. 175

540

P. TANDON and H. C. ARYA

Table 1. Auxin contents (ftY IAA equivalentlkg fresh weight) in in vivo and in vitro gaU and normal stem tissues.

in vivo tissuesl

Days

treatment in culture

5

10

15

20

25

30

41.2 ± 0.3 56.1 ± 0.3

32.4 ± 0.3 51.1 ± 0.3

26.1 ± 0.2 45.5 ± 0.2

21.5 ± 0.3 38.8 ± 0.2

17.2±0.2 31.4 ± 0.2

13.4 ± 0.1 21.5 ± 0.1

11.6 ± 0.1 19.7 ± 0.1

9.5 ± 0.1 18.0 ± 0.1

8.4 ± 0.1 14.8 ± 0.1

6.3 ± 0.1 12.0 ± 0.1

12.0 ± 0.1 17.6 ± 0.1

9.8 ± 0.1 16.5 ± 0.1

8.7 ± 0.1 15.1 ± 0.1

7.0 ± 0.1 13.0 ± 0.1

6.3 ± 0.1 10.5 ± 0.1

10.5 ± 0.1 16.5 ± 0.1

9.5 ± 0.1 14.8 ± 0.1

7.7 ± 0.1 13.7 ± 0.1

6.0 ± 0.1 11.6 ± 0.1

1n vivo tissues Normal 23.6 ± 0.3 42.0 ± 0.3 GaU DL-tryptophan Normal 3.5 ± 0.1 GaU 5.2 ± 0.1 NAA Normal 3.5 ± 0.05 GaU 5.2 ± 0.1 IAA Normal 3.5 ± 0.05 GaU 5.2 ± 0.1

5.2 ± 0.05 9.5 ± 0.1

± Standard error

tissues. The highest endogenous auxin level was found in in vivo normal and gall tissues followed by tissues grown in medium with DL-tryptophan (5 mg/I), NAA and IAA (10 mg/I), in the decreasing order. There are a number of reports on hyperauxin levels in crown-gall tissues (LOCKE et al. 1938; LINK and EGGERS 1941; KULESCHA and GAUTHERET 1948; NITSCH 1956). KEHR and SMITH (1954) reported that N. glauca x N. langsdorffii hybrids showed higher auxin content and a more effective enzyme system for converting tryptophan to auxin than the parents. BAYER (1965) demonstrated that the auxin content was highest in the hybrid, lower in N. glauca and lowest in N. langsdorffii. Similar results were obtained for N. longiflora, N. debneyi and their tumor producing hybrid (BAYER and AHUJA 1968). The potentially tumorous combinations showed higher levels of auxin than the parents and non tumorous segregunts. From the findings discussed above, it can be concluded that like crown-gallandgenetic tumors, Zizyphus gaU tissues showed auxin autotrophy and hyperauxinity. In both normal and gaU tissues grown in medium having auxin, the endogenous auxin level decreased. References AHUJA, M. R., and HAGEN, G. L.: Chromosomes and nutritional requirements of a tumor forming Nicotiana hybrid and its derivatives. (Abstr.) Am. J. Botany 53, 609 (1966). ARYA, H. C., VYAS, G. S., and TANDON, P.: The Problem of Tumor Formation in Plants. Form, Structure, and ~Function in Plants. (Ed. MOHAN RA)i, H. Y.; SHAH, J. J.; and SHAH, C. K.) pp. 270-279. Sarita Publishers, lIeerut, India 1975. BA YER, M. H.: Paper chrom<1tography of auxins and their inhibitors in two Nicotiana species and their hybrid. Am. J. Botany 52,883-890 (1965). and AHUJA, M. R.: Tumor formation in Nicotiana: Auxin levels and auxin inhibitors in normal and tumor prone genotypes. Planta 72. 292 - 298 (1968).

Auxin Autotrophy of Gall Tissue

541

BOUILLENNE, C., and GAS PAR, T.: Auxin catabolism and inhibitors in normal and crown-gall tissues of Impatiens balsam/:na. Can. J. Botany 48,1159-1163 (1970). BRAUN, A. C.: Abnormal growth in plants. Plant Physiology, a Treatise, Vo!. VB. (Ed. STEWARD, F. C.) pp. 379-420. Academic Press, New York 1969. - and STONIER, T.: Morphology and physiology of plant tumors. Protoplasmatologia 10,5 a. Springer Verlag, Vienna 1958. BUTCHER, D. N.: The origins, characteristics and culture of plant tumor cells. Plant Tissue and Cell Culture, Botanical Monographs vo!. 11. (Ed. STREET, H. E.) pp. 356-391. Blackwell Scientific Publications, Oxford, London, Edinburgh, }Ielbourne 1973. GAUTHERET, R. J.: Existe-t-il un eaneer des plantes? Universite de Paris. Conferenee au Palais de La Decouverte, Paris, Frame 1947. KEHR, A. E., and SlVlITH, H. H.: Genetic tumors in Nicoliana hybrids. Abnormal and Pathological Plants Growth. Brookhaven Symp. Biol. 6, 1954. KULEscHA, Z., and GAUTHERET, R. J.: Sur l'claboration de substances de croissance par 3 types, de cultures de tissus de Scorsonere: Cultures normales, Cultures de crown-gall et cultures accountumees a l'hetero-auxine. C. R. Acad. Sei. P,uis 227, 292 - 294 (1948). LINK, G. K. K., and EGGERS, V.: Hyperauxinity in erown-gaU of tomato. Botan. Gaz. 103, 87 -106 (1941). LOCKE, S. B., RIK EH, A. J., ami DUG(U R, B. }I.: Growth substanees and the development of erown-gaI!. J. Agr. Research 1)7,21-··89 (1938). MAHADEVAN, A.: Mcthods in Physiological Plant Pathology. 82p. Sivakami Publications, Madras, India 1975. MEINS, F. Jr.: Mechanisms underlying the persistence of tumor autonomy in crown-gall disease. Tissuc Culture aml Plant Seience. (Ed. STREET, H. E.) pp. 233-265. Proc. 3rd Intern. Congr. Plant Tissue and CeU Culture, Leieester, England. Academic Press, London and New York 1974. MURASHIGE, T., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobaceo tissue cultures. Physiol. Plantarum Vi, 473 - 479 (19G2). NITSCH, J. P.: Methods for the investigation of natnral auxins and growth inhibitors. The Chemistry and mode of action of plant growth substames (Eds. WAIN, R. L., and WIGIIT}L\N, F.) pp. 3-81. Butterworth, London and Washington, D.C. 195G. PELET, F., HILDEBRANDT, A. C., RIKEH, A. ,L, and SKOOG, F.: Growtll in vitra of tissues isolated from normal sterns ami insert gaUs. Am. J. Botany 47,186-195 (19GO). SCHAEFFER, G. W., and S:lIITH, H. H.: Auxin-kinetin intemction in tissue cllltures of Nicot/:ana species and tumor conditioned hybrids. Plant Physio!. 38, 291- 297 (1963). STONIER, T.: Studies on auxin protectors. VlI. Assoeiation of auxin protectors with crown-gaU development in sunflower sterns. Plant Physiol. 44, 11G9 -1174 (19G9). The role of auxin protettors in autonornous growth. Les Cultures de TissUR de Plantes. (Eds. HIRTH, M. L., amllVloREL, G.) pp. 42;3--4;35. Proe. Serond Intern. Conf. on Plant Tissue Cultllre, Strasbourg, Frame. Colloq. Intern. C.N.R.S., France 193, 1972. TANDON, P.: Further Studies on the Process of GaU Induetion on Zizyphus and the Factors Involved. Ph. D. Thesis, University of Jodhpur, Jodhpur, India 197G. - VYAS, G. S., anel AHY.I, IT. C.: ~Ie('hanism of in !:i/ra gall indllction in Zizyphus ,iujuba LAMK. Experientia 32,563-564 (1976). WHITE, P. R., and BHAt:N, A. C.: A t'tmcerous neoplasrn of plants. Antonornous bacteria-free crown-gall tissue. Cancer Research 2, 597 - Gl 7 (1942).

Received Decernber 8, 1979. Authors' addresses: Dr. PR,\J\lOD TAXDON, Department of Botany, School of Life Sciences, NorthEastern Hill University, Shillong 793014, India; Prof. Dr. H. C. ARYA, Department of Botany, Jodhpur University, Jodhpur 342001, India. 37*